Stabilization of Myc through Heterotypic Poly-Ubiquitination by mLANA Is Critical for γ-Herpesvirus Lymphoproliferation

Host colonization by lymphotropic γ-herpesviruses depends critically on expansion of viral genomes in germinal center (GC) B-cells. Myc is essential for the formation and maintenance of GCs. Yet, the role of Myc in the pathogenesis of γ-herpesviruses is still largely unknown. In this study, Myc was shown to be essential for the lymphotropic γ-herpesvirus MuHV-4 biology as infected cells exhibited increased expression of Myc signature genes and the virus was unable to expand in Myc defficient GC B-cells. We describe a novel strategy of a viral protein activating Myc through increased protein stability resulting in increased progression through the cell cycle. This is acomplished by modulating a physiological post-translational regulatory pathway of Myc. The molecular mechanism involves Myc heterotypic poly-ubiquitination mediated via the viral E3 ubiquitin-ligase mLANA protein. EC5SmLANA modulates cellular control of Myc turnover by antagonizing SCFFbw7 mediated proteasomal degradation of Myc, mimicking SCFβ-TrCP. The findings here reported reveal that modulation of Myc is essential for γ-herpesvirus persistent infection, establishing a link between virus induced lymphoproliferation and disease

Murine Gammaherpesvirus 68 LANA Acts on Terminal Repeat DNA To Mediate Episome Persistence

Murine gammaherpesvirus 68 (MHV68) ORF73 (mLANA) has sequence homology to Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). LANA acts on the KSHV terminal repeat (TR) elements to mediate KSHV episome maintenance. Disruption of mLANA expression severely reduces the ability of MHV68 to establish latent infection in mice, consistent with the possibility that mLANA mediates episome persistence. Here we assess the roles of mLANA and MHV68 TR (mTR) elements in episome persistence. mTR-associated DNA persisted as an episome in latently MHV68-infected tumor cells, demonstrating that the mTR elements can serve as a cis-acting element for MHV68 episome maintenance. In some cases, both control vector and mTR-associated DNAs integrated into MHV68 episomal genomes. Therefore, we also assessed the roles of mTRs as well as mLANA in the absence of infection. DNA containing both mLANA and mTRs in cis persisted as an episome in murine A20 or MEF cells. In contrast, mTR DNA never persisted as an episome in the absence of mLANA. mLANA levels were increased when mLANA was expressed from its native promoters, and episome maintenance was more efficient with higher mLANA levels. Increased numbers of mTRs conferred more efficient episome maintenance, since DNA containing mLANA and eight mTR elements persisted more efficiently in A20 cells than did DNA with mLANA and two or four mTRs. Similar to KSHV LANA, mLANA broadly associated with mitotic chromosomes but relocalized to concentrated dots in the presence of episomes. Therefore, mLANA acts on mTR elements to mediate MHV68 episome persistence

Effects of Dry Matter Content and Microbial Additive on Tifton 85 \u3ci\u3e(Cynodon dactylon ssp.)\u3c/i\u3e Wilted Silage Fermentation Parameters

The objective of this study was to evaluate the wilting and the addition of a bacterial-enzymatic additive effects on the fermentation parameters of Tifton 85 (Cynodon dactylon spp.) silage. Forage was stored as 326 kg bales wrapped with a plastic film. Treatments consisted of 5 forage dry matter levels (20-30%, 30-40%, 40-50%, 50 -60% e 60 a 70%) without additive and 3 dry matter levels (20-30%, 40-50%, e 60-70%) with additive. Buffered propionic acid solution was sprayed onto 60-70% dry matter bales, prior to wrapping, determining an additional treatment. Core samples were taken at 0, 6, 12 hours and 1, 2, 4, 8, 16, and 32 days after wrapping to establish silage pH and temperature trends. Field dry matter losses during the baling process were also evaluated. Bale weight with no additive decreased (364 kg to 254 kg) with increased forage DM content, which in turn resulted in lower bale bulk density (310 to 216 kg/m3 ). Lower field DM losses (281 to 177 kg/ha) were associated with higher forage DM content. Final silage pH and temperature peaks were increased at higher DM content, whereas the presence of microbial additive prevented temperature surge

Neural Online Filtering Based on Preprocessed Calorimeter Data

Aiming at coping with LHC high event rate, the ATLAS collaboration has been designing a sophisticated three-level online triggering system. A significant number of interesting events decays into electrons, which have to be identified from a huge background noise. This work proposes a high-efficient L2 electron / jet discrimination algorithm based on artificial neural processing fed from preprocessed calorimeter information. The feature extraction part of the proposed system provides a ring structure for data description. Energy normalization is later applied to the rings, making the proposed system usable for a broad energy spectrum. Envisaging data compaction, Principal Component Analysis and Principal Component of Discrimination are compared in terms of both compaction rates and classification efficiency. For the pattern recognition section, an artificial neural network was employed. The proposed algorithm was able to achieve an electron detection efficiency of 96% for a false alarm of 7%

GLUT1-mediated glucose uptake plays a crucial role during Plasmodium hepatic infection.

Intracellular pathogens have evolved mechanisms to ensure their survival and development inside their host cells. Here, we show that glucose is a pivotal modulator of hepatic infection by the rodent malaria parasite Plasmodium berghei and that glucose uptake via the GLUT1 transporter is specifically enhanced in P. berghei-infected cells. We further show that ATP levels of cells containing developing parasites are decreased, which is known to enhance membrane GLUT1 activity. In addition, GLUT1 molecules are translocated to the membrane of the hepatic cell, increasing glucose uptake at later stages of infection. Chemical inhibition of GLUT1 activity leads to a decrease in glucose uptake and the consequent impairment of hepatic infection, both in vitro and in vivo. Our results reveal that changes in GLUT1 conformation and cellular localization seem to be part of an adaptive host response to maintain adequate cellular nutrition and energy levels, ensuring host cell survival and supporting P. berghei hepatic development

Framing Hospital Engagement for the Recruitment of a Birth Cohort for the NCS: Lessons Learned for Ensuring Collaboration in Worcester County

In 2011, three designated NCS Study Centers began preparatory work for field implementation of a planned recruitment strategy called Provider Based Sampling (PBS). In each PBS primary sampling unit, three hospitals were selected to test the feasibility of recruiting a cohort of 125 women and their babies around delivery time. The selected hospitals for Worcester account for nearly 80% of County births and can be categorized into three distinct facility types and patient catchment areas: an academic medical center; a university-affiliated but independent community hospital; and a private for-profit community hospital with market share competitor of the academic medical center. Methods: We used tailored negotiations and engagement strategies to gain the cooperation and engagement of targeted hospitals/birthing centers. Preliminary Conclusions: The lessons learned from this exercise are:• Time to gain hospital engagement and clearance to initiate study activities ranges anywhere from 2 weeks to 2 months and depends largely upon the type of the institution, the profile of the Negotiator, and the nature of the scope of work.• A greater likelihood of hospital engagement in the NCS seems to be associated with the depth of existing relationships between the Study Center and targeted hospitals.• Thoughtful interactions and timely discussions with the key institutional stakeholders (either individually or in groups) are important to achieve collaboration and engagement.• Balancing sensitivity to clinical cultures and settings while preserving research integrity is essential for study implementation in busy hospital/clinical environments.• Planning for site compensation and/or the ability to support local clerical staff to help with study activities must be considered as a means to facilitate negotiations and site engagement.• Adequate resources must be planned for successful implementation and execution of research activities in settings (e.g community hospitals) unfamiliar with research activities.• Involvement of nursing personnel is crucial for successful implementation of any protocol

A Comparison of Recruitment Strategies for a Long-Term Study at Two Maternal Stages: Effectiveness of Recruitment During Pregnancy vs. After Childbirth

Introduction. National Children’s Study (NCS) Provider Based Sampling (PBS) aims to conduct a pilot study to test cost, acceptability and feasibility of recruiting a representative sample of women/children using two recruitment strategies: through prenatal providers and hospitals. Methods. A sampling frame consisting of all providers of prenatal and delivery care within and 10-miles outside Worcester County, 16 provider and 3 hospital locations were selected as point of entry for study recruitment. During 1st prenatal care visits or post-delivery at these locations, face-to-face contact was utilized to: a) identify study eligibility and b) assess study recruitment. Preliminary Results. Certified Data Collectors made contact with prescreened women. Consent rates of women at prenatal provider locations were lower than the consent rates in hospital locations. On average, results have shown twice as many consents could be obtained per day at hospital locations than at provider locations. Preliminary Conclusions. Although both strategies utilized direct rapport, the two recruitment methods were associated with different consent rates. Consideration of preliminary results may lead one to consider recruitment after childbirth for several reasons: 1) greater likelihood of having opportunity to discuss study with the woman and partner from outset; 2) opportunity to check back with undecided women easily 3) longer periods to answer questions and conduct screening and consent; 4) support of nursing staff to foster participation; 5) daily presence of NCS staff; and 6) reality of infant’s birth to spur mother to consent. Recruitment during pregnancy visits may yield lower rates; further examination may be necessary to overcome challenges such as: 1) burden of adding recruitment session to often long and anxiety-laden1st prenatal visit; 2) need to develop rapport quickly during brief time periods; 3) making contact with potential participants outside of provider office when recruitment is not completed